Immunohistochemical studies on phosphorylation of tyrosine hydroxylase in central catecholamine neurons using site- and phosphorylation state-specific antibodies.
Immunohistochemical studies on phosphorylation of tyrosine hydroxylase in central catecholamine neurons using site- and phosphorylation state-specific antibodies.
复制标题
使用位点和磷酸化状态特异性抗体对中枢儿茶酚胺神经元中酪氨酸羟化酶的磷酸化进行免疫组织化学研究。
DOI：
10.1016/s0306-4522(97)00189-9
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发表时间：
1998
期刊：
影响因子：
3.3
通讯作者：
Hökfelt,T
中科院分区：
文献类型：
--
作者：
Xu,ZQ;Lew,JY;Harada,K;Aman,K;Goldstein,M;Deutch,A;Haycock,JW;Hökfelt,T
酪氨酸羟化酶的磷酸化形式，第一个和速率限制酶的儿茶酚胺生物合成的抗体，被施加在免疫组织化学研究大鼠脑片体外孵育与磷酸二酯酶抑制剂（3-异丁基-1-甲基黄嘌呤，IBMX）和福司可林福尔马林灌注大鼠大脑。使用了四种抗血清/抗体：（i）丝氨酸40处磷酸化的酪氨酸羟化酶（THS 40 p抗血清）、（ii）丝氨酸19处磷酸化的酪氨酸羟化酶（THS 19 p抗血清）、（iii）天然酶（泛酪氨酸羟化酶抗血清）的多克隆兔抗血清和（iv）天然酪氨酸羟化酶的小鼠单克隆抗体。在体外研究中，没有观察到THS 40 p样免疫反应性，除非切片用IBMX-毛喉素处理，之后在纹状体中发现致密的纤维网络，并且在腹侧中脑中发现免疫反应性细胞体，特别是在腹侧被盖区。虽然这些细胞是泛酪氨酸羟化酶阳性，其中几个没有染色的酪氨酸羟化酶单克隆抗体。此外，有一个显着的减少酪氨酸羟化酶单克隆抗体免疫反应性纤维在药物处理的切片，这表明酪氨酸羟化酶单克隆抗体不承认丝氨酸40磷酸化形式的酪氨酸羟化酶。在多巴胺能A11细胞组中，处理过的切片没有显示任何THS 40 p免疫反应阳性细胞体，在蓝斑中仅观察到少数弱荧光神经元。然而，在蓝斑中观察到稀疏的纤维lexus，可能反映肾上腺素纤维。在灌注的大脑中，THS 40 p样免疫反应可以可视化在一些多巴胺神经元在腹侧中脑，特别是A10区，和去甲肾上腺素能蓝斑神经元，而THS 19 p样免疫反应被发现在所有的儿茶酚胺组研究，类似的结果与泛酪氨酸羟化酶抗血清和酪氨酸羟化酶单克隆抗体。在前脑区已知的中脑多巴胺神经元支配，没有THS 40 p阳性纤维观察到，而THS 19 p免疫反应性纤维被发现在纹状体，嗅结节和神经核的子区域，基本上重叠与多巴胺纤维先前显示含有胆囊收缩素样免疫反应。目前的结果表明，针对磷酸化形式的酪氨酸羟化酶的抗体可用于评估酪氨酸羟化酶的磷酸化状态在个别神经元细胞体和过程在体外和体内。
Antibodies raised to phosphorylated forms of tyrosine hydroxylase, the first and rate-limiting enzyme in the catecholamine biosynthesis, were applied in immunohistochemical studies on rat brain slices incubated in vitro with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) and forskolin on formalin-perfused rat brains. Four antisera/antibodies were used: polyclonal rabbit antisera to (i) tyrosine hydroxylase phosphorylated at serine 40 (THS40p antiserum), (ii) tyrosine hydroxylase phosphorylated at serine 19 (THS19p antiserum), (iii) to the native enzyme (pan-tyrosine hydroxylase antiserum), and mouse monoclonal antibody to (iv) native tyrosine hydroxylase. In the in vitro studies THS40p-like immunoreactivity was not observed unless slices were treated with IBMX–forskolin after which a dense fibre network was found in the striatum, and immunoreactive cell bodies were found in the ventral mesencephalon, especially in the ventral tegmental area. Although these cells were pan-tyrosine hydroxylase-positive, several of them were not stained with the tyrosine hydroxylase-monoclonal antibody. Moreover, there was a marked reduction of tyrosine hydroxylase-monoclonal antibody-immunoreactive fibres in drug-treated slices, suggesting that this tyrosine hydroxylase-monoclonal antibody does not recognize the Serine 40-phosphorylated form of tyrosine hydroxylase. Treated slices did not show any THS40p-immunoreactive cell bodies in the dopaminergic A11 cell group and only a few, weakly fluorescent neurons were observed in locus coeruleus. However, a sparse fibre lexus was observed in locus coeruleus, possibly reflecting epinephrine fibres. In the perfused brains THS40p-like immunoreactivity could be visualized in some dopamine neurons in the ventral mesencephalon, especially the A10 area, and in noradrenergic locus coeruleus neurons, whereas THS19p-like immunoreactivity was found in all catecholamine groups studied, similar to the results obtained with the pan-tyrosine hydroxylase antiserum and the tyrosine hydroxylase-monoclonal antibody. In forebrain areas known to be innervated by mesencephalic dopamine neurons, no THS40p-positive fibres were observed, whereas THS19p-immunoreactive fibres were found in subregions of the striatum, olfactory tubercle and nucleus accumbens, essentially overlapping with dopamine fibres previously shown to contain cholecystokinin-like immunoreactivity. The present results suggest that antibodies directed against phosphorylated forms of tyrosine hydroxylase can be used to evaluate the state of tyrosine hydroxylase phosphorylation in individual neuronal cell bodies and processes both in vitro and in vivo.